Abstract Insulin resistance is central to the pathogenesis of Type 2 Diabetes (T2DM) and the Metabolic Syndrome, and drugs or lifestyle interventions that increase insulin sensitivity constitute effective therapy and prevention. Thiazolidinedione drugs (TZDs) act through agonism of nuclear transcription factors (i.e., PPAR) to enhance insulin sensitivity; however, TZD therapy is associated with adverse effects, including weight gain and heart failure. These untoward effects limit clinical utility, and highlight the need for alternative insulin-sensitizing medications that can act directly on muscle. We will study the NR4A family of orphan nuclear receptors, which were identified as differentially expressed genes on our human muscle cDNA microarrays. Extensive preliminary data indicate that: (i) NR4A3 is expressed at lower levels in insulin-resistant humans and rodents; (ii) TZDs induce NR4A3 suggesting that NR4A3 may be downstream of TZD action; (iii) MCK-NR4A3 transgenic mice exhibit an insulin sensitive phenotype; (iv) an increase in NR4A3 receptors can augment insulin signaling and stimulation of glucose transport; (v) PGA2 acts to increase insulin sensitivity in a NR4A3 dependent manner. To pursue these novel observations, our overall goal is to increase our understanding of the role of NR4A receptors in modulating insulin action, their role in human insulin resistance, and their rationale as a therapeutic drug target. To achieve this goal, we will apply our laboratory's capacity for translational research including human metabolism, human muscle and adipose tissue biopsies, transgenic mice, and cultured cell systems. Based on extensive preliminary data, the specific aims are: (1) Assess expression of NR4A receptors in muscle and fat in insulin sensitive, insulin resistant, and T2DM humans, before and after weight loss and TZD treatment, and in insulin resistant rodent models. (2) Establish metabolic role of NR4A3 by phenotyping transgenic mice with specific hyperexpression of NR4A3 in skeletal muscle. In both human and mouse, NR4A expression will be assessed for its ability to affect insulin sensitivity and substrate metabolism at the level of whole body and individual cells and tissues. (3) Determine mechanisms by which NR4A3 regulates insulin action by studying stable hyperexpression and shRNA- mediated suppression of NR4A3 in cultured muscle and adipose cells. These studies will address our preliminary data indicating that NR4A3 modulates insulin-stimulated glucose transport, GLUT4 translocation, and insulin-mediated phosphorylation of signaling molecules. (4) Identify lipid mediator agonists of NR4A3 based on preliminary data indicating that PGA2 increases insulin sensitivity in a NR4A3 dependent manner. Thus, this work will elucidate novel molecules and pathophysiologic processes contributing to insulin resistance, and develop new potential drug targets for the treatment and prevention of diabetes and cardiometabolic disease.